Assays have been developed for detecting and quantifying the presence of substances of interest using immunological binding. Such assays include those which use antibodies which recognize a region or "epitope" on an antigenic molecule, responsible for the specific interaction of the antigen with the antibody. Of particular interest are assays capable of detecting substances in a fluid sample such as blood serum, for example, certain glycoproteins and glycolipids which have been found to be associated with tumor tissues and may thus serve as tumor markers. Recently, a class of high molecular weight glycoproteins known as mucins, containing large amounts of carbohydrates, has been found to be tumor-associated. Monoclonal antibodies capable of detecting epitopes on mucin antigens have been described. Immunoassays using such monoclonal antibodies have shown promise for the detection and monitoring of cancer in humans. These antibodies reactive with mucins include CA 19-9, (Koprowski et al., Science, 212:53-54 (1981)); CA 50, (Holmgren et al., Br. Med. J. 280:1479-82 (1984)); DUPAN-2, (Metzgar et al., PNAS 81:5242-5246 (1984)); and antibody to sialylated Le.sup.x epitope (Kannagi et al., Cancer Res. 46:2619-2626 (1986)).
Sialic acids, which are O-acyl derivative of N-acetyl neuraminic acid, an amino sugar acid, are widely distributed in human tissues as constituents of lipids, polysaccharides and of mucoproteins. Sialic acid occurs in various forms which differ in their acyl side chains. Sialic acids containing O-acetyl, O-methyl and O-glycolyl constituents have been described.
Tumor cells have been found to contain relatively large amounts of sialic acid, and mucin antigens present in serum derived from cancer patients have been reported to be sialylated. The enzyme neuraminidase removes sialic acid. In some cases, it has been observed that the ability of a monoclonal antibody to bind with an epitope on a mucin antigen normally recognized by the antibody is decreased after digestion of the antigen using neuraminidase. (Kannagi et al., supra). It has also been observed that neuraminidase treatment can render tumor associated glycoproteins more immunologically reactive (U.S. Pat. No. 4,146,603). Based on these observations, neuraminidase sensitivity has been used to assist in the characterization of epitopes recognized by various antibodies.
The resulting decrease in the ability of "neuraminidase sensitive" antibodies to bind to neuraminidase treated sialylated antigen has been explained by suggesting that sialic acid is required for proper three-dimensional conformation of these binding sites. (Johnson et al., Cancer Res., 46:850-857 (1986)). Other sialylated tumor-associated antigens include the Lewis blood group antigens and carcinoembryonic antigen (CEA) (Kannagi et al., supra).
Because sialic acid is generally found as a terminal residue in oligosaccharide side chains, it may also block or "mask" epitopes. For example, lectin binding sites in gastric tumors are exposed following removal of sialic acid using neuraminidase (Macartney, J. Pathol. 150:135-144 (1986)). In addition to sialic acid, other peripheral sugars such as fucose also interfere with immunological binding sites on ligands such as antigens. The removal of such peripheral substances may reveal "core" carbohydrates in proteins (Feize et al., Biochem. Soc. Trans. 12:591-599 (1984)) which contain binding sites not otherwise exposed. These core structures are common in oligosaccharide chains from many sources, and, in general, do not exhibit the restricted tissue distribution of peripheral oligosaccharide structures such as the Lewis blood group antigens.
Previous methods for removing peripheral sugars from proteins have required purification of ligand prior to treatment. It would thus be beneficial to develop a method for removing substances from ligands such as antigens without prior purification to make additional immunological binding sites available to serve as improved disease or infection markers.